Enitociclib, a Selective CDK9 Inhibitor, Induces Complete Regression of MYC+ Lymphoma by Downregulation of RNA Polymerase II Mediated Transcription

Abstract Double-hit diffuse large B-cell lymphoma (DH-DLBCL) is an aggressive, and often refractory, type of B-cell non–Hodgkin lymphoma (NHL) characterized by rearrangements in MYC and BCL2. Cyclin-dependent kinase 9 (CDK9) regulates transcriptional elongation and activation of transcription factors, including MYC, making it a potential targeted approach for the treatment of MYC+ lymphomas. Enitociclib is a well-tolerated and clinically active CDK9 inhibitor leading to complete metabolic remissions in 2 of 7 patients with DH-DLBCL treated with once weekly 30 mg intravenous administration. Herein, we investigate the pharmacodynamic effect of CDK9 inhibition in preclinical models and in blood samples from patients [DH-DLBCL (n = 10) and MYC+ NHL (n = 5)] treated with 30 mg i.v. once weekly enitociclib. Enitociclib shows significant regulation of RNA polymerase II Ser2 phosphorylation in a MYC-amplified SU-DHL-4 cell line and depletion of MYC and antiapoptosis protein MCL1 in SU-DHL-4 and MYC-overexpressing SU-DHL-10 cell lines in vitro. Tumor growth inhibition reaching 0.5% of control treated SU-DHL-10 xenografts is achieved in vivo and MYC and MCL1 depletion as well as evidence of apoptosis activation after enitociclib treatment is demonstrated. An unbiased analysis of the genes affected by CDK9 inhibition in both cell lines demonstrates that RNA polymerase II and transcription pathways are primarily affected and novel enitociclib targets such as PHF23 and TP53RK are discovered. These findings are recapitulated in blood samples from enitociclib-treated patients; while MYC downregulation is most robust with enitociclib treatment, other CDK9-regulated targets may be MYC independent delivering a transcriptional downregulation via RNA polymerase II. Significance: MYC+ lymphomas are refractory to standard of care and novel treatments that downregulate MYC are needed. The utility of enitociclib, a selective CDK9 inhibitor in this patient population, is demonstrated in preclinical models and patients. Enitociclib inhibits RNA polymerase II function conferring a transcriptional shift and depletion of MYC and MCL1. Enitociclib intermittent dosing downregulates transcription factors including MYC, providing a therapeutic window for durable responses in patients with MYC+ lymphoma.


Introduction
Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL) subtype (1).Although standard immunochemotherapy often cures patients with DLBCL (2,3), up to 40% of patients will eventually relapse (4,5).Many of these patients are diagnosed with high-grade B-cell refs.16,17) and in turn is required for the elongation of mRNA transcripts.
MYC is a pleiotropic transcription factor, which controls cell cycle, DNA damage repair, and cell metabolism by regulating numerous target genes (18).
MYC mRNA has a short half-life; therefore, high rates of MYC transcription are necessary to drive oncogenic signaling (19)(20)(21).Myc protein expression is consequently dependent on CDK9 phosphorylation of p-Ser2.Mcl-1 is an antiapoptotic protein belonging to Bcl-2 family (22).Like Bcl-2, Mcl-1 promotes cell survival (23) and many hematologic malignancies are dependent on the antiapoptotic activity of Bcl-2 or Mcl1 (24).Mcl-1 has been implicated as an apoptotic regulator in Eμ-Myc-driven lymphomas and is regulated by P-TEFb (25).Dinaciclib, a pan-CDK inhibitor, has been shown to downregulate MCL1 leading to apoptosis induction and antitumor efficacy in DLBCL preclinical models (25) and clinical activity has been demonstrated in patients with relapsed/refractory chronic lymphocytic leukemia (CLL; ref. 26).
Enitociclib, formerly known as VIP152, is a well-tolerated and clinically active CDK9 inhibitor that has led to durable complete metabolic remission (CR) in 2 of 7 patients with DH-DLBCL treated once weekly with 30 mg enitociclib by intravenous infusions (27,28).The pharmacodynamic effect of in blood of patients treated with enitociclib at dose levels lower than 30 mg demonstrates that MYC, MCL, and PCNA mRNA downregulation is not as robust and/or durable (29).Enitociclib is administered intravenously once weekly to allow for on-target Mcl-1 downregulation in neutrophils to recover before the next dose providing a wider therapeutic window unlike other orally administered CDK9 inhibitors in development such as atuveciclib and KB-0742 (30,31).Previous studies using an acute myeloid leukemia (AML) xenograft model have demonstrated excellent in vivo efficacy of enitociclib (32).Herein, we elucidate the in vitro mechanism of action of enitociclib, atuveciclib, and KB-0742 in MYCdriven DLBCL preclinical models and demonstrate that enitociclib treatment induces a depletion of MYC as well as describe novel CDK9 inhibitor target genes that are downregulated on a different time course to deliver an "oncogenic shock" (33,34) in cells addicted to MYC expression.These findings are confirmed in the blood of patients with MYC+ lymphoma treated with enitociclib and elucidate the mechanism of action of P-TEFb targeting by selective CDK9 inhibition for the treatment of patients with NHL with MYC-driven disease.

Compounds and Cell Lines
Human DLBCL cell lines SU-DHL-4 (#ACC 495) and SU-DHL-10 (#ACC 576) were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures).The supplier used PCR-based DNA profiling of polymorphic short tandem repeats for authentication and Mycoplasma testing prior to shipment.Cells were cultured in suspension at 37°C in a humidified atmosphere containing 5% CO 2 .CDK9 inhibitors KB-0742 (MedChemExpress, #HY-137478A), enitociclib, and atuveciclib (Vincerx Pharma, Inc.) were used.A subset of 35 lymphoma cell lines is from a 500-cell line panel where the effect of enitociclib on cell viability using CellTiter-Glo luminescent cell viability was determined (OmniScreen, CrownBioscience).Genetic status and gene expression level for MYC and MCL was obtained from CrownBioscience.

RNA-seq and qPCR
Total RNA was extracted from blood of patients and cell lines using RNeasy Plus Mini Kit (QIAGEN, #74136) and cDNA prepared with the Maxima First Strand cDNA Synthesis Kit for RT-PCR (Thermo Fisher Scientific, K1642) according to manufacturer's instructions.The integrity of purified total RNA was evaluated using an Agilent 2100 Bioanalyzer system and RNA 6000 Nano Kit (Agilent, #5067-1511).Differential gene expression analysis was performed (Fios Genomics) from RNA-seq data (Discovery Life Sciences).MYC and MCL mRNA levels were measured by qPCR in vitro and in vivo, using a Taqman qPCR system (Thermo Fisher Scientific), TaqMan Fast Universal PCR Master Mix (2X) no AmpErase (#4352042) and TaqMan probes for MYC (Hs00153408_m1) and MCL (Hs03043899_m1).RNA-seq data were analyzed using DESeq2 (35).

Animal Experiments
A total of 10 × 10 6 SU-DHL-10 cells were inoculated subcutaneously into 6-8 weeks old female C.B-17 SCID mice (Iffa Credo, Brussels).The mice were randomized to treatment groups at a tumor volume of 63-104 mm 3 and treated (intravenously, once weekly) with 10 mg/kg enitociclib (60% PEG400, 10% ethanol, water), 15 mg/kg enitociclib (30% PEG400, 10% ethanol, water), or corresponding vehicle treatments.Mice were around 10-12 weeks of age at the start of study.All animal experiments were performed under the national animal welfare laws of Germany and approved by the local authorities.
Statistical analyses were performed using GraphPad Prism v9 (GraphPad Software).For tumor volumes in SU-DHL-10 xenograft model, statistics were performed using unpaired t test in comparison with the corresponding vehicle treatment.The obtained P values were adjusted for all analyses.Results were considered statistically significant when P < 0.05.

Pharmacokinetic and Pharmacodynamic Modeling of Patient Samples
All pharmacokinetic parameters were calculated by noncompartmental methods as described previously (36)

Differential Gene Expression Analysis
RNA-seq data generated from the analysis of two cell lines treated with the three CDK9 inhibitors was analyzed to investigate the differences in gene expression levels between various posttreatment timepoints and the pretreatment samples.Quality control assessment and exploratory data analysis by principal component analysis (PCA) were done on raw counts and on normalized count data.Normalization of RNA-seq count data was done using trimmed mean of M-values normalization and transformed with voom, resulting in log 2 counts per million with associated precision weights.To determine significantly differentially expressed genes (DEG) between different timepoints of treatment with DMSO (control), CDK9 inhibitor treatment and the pretreatment control samples, statistical comparisons were performed using linear modeling on normalized data as implemented in the Bioconductor package limma, and the final values were expressed as a percentage of baseline.Significance values (P adj ) were adjusted for multiple testing, by controlling the FDR using the Benjamini-Hochberg procedure.Significant DEGs (at P adj ≤ 0.05, fold change ≥2) identified were then analyzed for enrichment of Reactome pathways using a hypergeometric test, and the enrichment (P < 0.05) was assessed together for the upregulated and downregulated genes.RNA-seq data generated from blood samples taken from patients before and after their first dose (C1D1) or after multiple doses, before and after their third dose (C1D15) were analyzed and normalized using the same pipeline as cell lines for comparisons.

Enitociclib Delivers Robust Inhibition of RNA Polymerase II Ser2 Phosphorylation for up to 48 Hours in Cell Lines
Enitociclib induced cell cytotoxicity (IC 50 values ranged from 0.043 to 0.152 μmol/L) in a panel of 35 human lymphoma cell lines.MYC and MCL mRNA downregulation has been described previously from the blood of patients treated with enitociclib during dose escalation and across many cancer indications including NHL and solid tumors using a qPCR clinical trial assay (28,29).Because these genes are thought to drive tumorigenesis, the genetic status and expression level of MYC and MCL for each line is indicated (25); however, the correlations between these aberrations and enitociclib sensitivity and resistance were not possible as most lymphoma cell lines are sensitive to enitociclib treatment (Supplementary Table S1).out and the pharmacodynamic effect was observed at the indicated timepoints (Fig. 1B).In the SU-DHL-4 cell line, treatment with 0.25 μmol/L enitociclib decreased the levels of p-Ser2 by 50% for up to 48 hours.The extent of p-Ser2 inhibition was greater at the higher 1 μmol/L enitociclib treatment, sustaining for 48 hours.In contrast, treatment with 1 μmol/L KB-0742 elicited a 50% decrease for up to 12 hours in alignment with a previous report using a MYC-driven AML xenograft mouse model (31).Treatment with 1 μmol/L atuveciclib approached 50% inhibition of p-Ser2 for up to 16 hours (Fig. 1C and D), a marker known to be downregulated by atuveciclib in T-cell prolymphocytic leukemia models (38).In the SU-DHL-10 cell line, 1 μmol/L enitociclib was the only treatment able to achieve 50% reduction of p-Ser2 for up to 24 hours after treatment.Atuveciclib and KB-0742 also inhibited p-Ser2, but only in the presence of the treatment compound and not after washout (Fig. 1D).Comparison of each drug treatment demonstrates that the p-Ser2 changes on RNA polymerase II are statistically significant in the SU-DHL-4 cell line at all timepoints (Supplementary Table S2).The limitation of the experimental design is that the exposure of KB-0742 may not mimic the clinical pharmacokinetic profile.These are not public at time of writing therefore previously published concentration of 1 μmol/L was used in these studies (31).

Enitociclib Depletes mRNA transcripts, MYC and MCL1; Downregulates Myc and Mcl1 Protein Levels and Activates Cell Death In Vitro
Because CDK9 inhibition abrogates transcription elongation of mRNAs by RNA polymerase II, we next evaluated the extent and duration of the effect of CDK9 inhibition on mRNA levels of MYC and MCL by qPCR.MYC and MCL mRNA levels were decreased with all three CDK9 inhibitors initially; however, only atuveciclib and enitociclib were able to drive the depletion of MYC (Fig. 2A) and MCL (Fig. 2B) mRNA transcript after treatment washout in SU-DHL-10 and SU-DHL-4 cell lines.In SU-DHL-10 cells, atuveciclib and KB-0742 were able to maintain approximately 50% depletion of the MYC and MCL mRNA levels for up to 16 hours after treatment, whereas enitociclib inhibited MYC and MCL mRNA levels approximately 75% for 16 to 48 hours after treatment.In MYC-amplified SU-DHL-4 cells, only 1 μmol/L enitociclib maintained durable depletion of MYC and MCL mRNA levels over the 48-hour time course (Fig. 2A and B).At the protein level, 1 μmol/L enitociclib was the only treatment that drove sustained Myc (Fig. 2C) and Mcl-1 (Fig. 2D) protein depletion for 48 hours in the SU-DHL-10 cells; whereas this was achieved in SU-DHL-4 cells with 0.25 μmol/L enitociclib and atuveciclib treatments.Protein levels of cPARP, a marker for apoptosis, were 3-to 5-fold higher at peak occurring 24 hours after treatment washout in SU-DHL-10 cells treated with enitociclib or atuveciclib in comparison with KB-0742, which had no effect on cPARP (Fig. 2E).These results suggest that sustained in vitro control of Myc protein levels and activation of cell death in MYC+ lymphoma cell line models are driven by enitociclib in a dose-dependent manner.

Enitociclib Treatment Results in Complete Regression of MYC-overexpressing SU-DHL-10 Lymphoma Growth and Mechanism of Action Confirmed In Vivo
The antitumor efficacy of enitociclib was evaluated in the SU-DHL-10 in vivo xenograft mouse model.A dose-dependent antitumor efficacy was observed, resulting in SU-DHL-10 tumor growth control with 10 mg/kg enitociclib.
Whereas 15 mg/kg enitociclib induced complete regression during the 3-week once weekly treatment period (Fig. 3A).On days 16 and 20 after treatment start, the treatment/control (T/C) values for 10 and 15 mg/kg enitociclib were 0.19 (P = 0.011) and 0.005 (P < 0.001), respectively (Fig. 3A).Subsequently, tumors in mice treated with 15 mg/kg enitociclib were allowed to regrow until a new treatment cycle with enitociclib (15 mg/kg, once weekly) was introduced on study day 29 again leading to regression of tumors (Fig. 3A).Changes in body weights of treated mice were observed during the study where recovery is observed before the next weekly dose and the maximum body weight change observed was −12.5% at 15 mg/kg (Fig. 3B).The pharmacodynamic effect of enitociclib in the tumors of SU-DHL-10 in vivo xenograft mouse model was evaluated after a single dose of 5, 10, or 15 mg/kg.Treatment with 5 mg/kg enitociclib decreased the levels of p-Ser2 by greater than 50% for 8 hours; whereas the 10 and 15 mg/kg doses resulted in depletion of p-Ser2 in the same timeframe postdose.In contrast, p-Ser5 reductions at all doses were not robust (Fig. 3C).
A dose-dependent depletion of MYC mRNA detected by qPCR was observed.At the 10 and 15 mg/kg doses, MYC mRNA is depleted by 4 hours postdose.
Apoptotic regulator MCL mRNA levels were also reduced by 4 hours postdose, and depletion was sustained for 8 hours postdose at the two higher dose levels.(Fig. 3D).Myc protein levels were reduced by 50% and Mcl-1 protein levels robustly decreased within 4 to 8 hours postdose.Activation of apoptosis was observed as measured by an increase in cleaved PARP (Fig. 3E).

Enitociclib Treatment Confers a Robust Shift in Transcriptional Activity in MYC+ DLBCL Cell Lines
Given that the P-TEFb complex regulates RNA polymerase II transcription via CDK9 kinase activity, the effects of three CDK9 inhibitors, enitociclib, atuveci-clib, and KB-0742 on gene expression in SU-DHL-4-and SU-DHL-10-treated cell lines (Fig. 1B) was assessed by RNA-seq.According to PCA, the transcriptional profiles are most variable based on their cell line of origin and then the variance in the raw data is explained mostly by timepoint (Supplementary Fig. S1A).RNA-seq profiles from both cell lines were compared with each other for each treatment as well as compared with DMSO treatment and parental cell lines.A heat map of 1,491 significantly DEGs after 4 hours 250 nmol/L enitociclib treatment demonstrates gene intensity per sample relative to the average level across all samples for all treatments.Unsupervised clustering of samples demonstrates that enitociclib treatment (250 nm and 1 μmol/L) and 1 μmol/L atuveciclib cluster together while those samples treated with DMSO or 1 μmol/L KB-0742 cluster together.The majority of DEGs are downregulated (1,177/1,491; 78.94%) which drives the clustering of genes into two branches of the dendrogram (Fig. 4A).The top DEGs from RNA-seq analysis of samples from cell lines treated with each of the four treatments are listed in order of their P adj < e-7 and unique or common DEGs are color coded (Fig. 4B), and fold change and P values are listed (Supplementary Table S3).Three of the four identified pathways in enitociclib and atuveciclib 1 μmol/L treatments were the same, where generic transcription, RNA polymerase II transcription and gene expression (Transcription) pathways were statistically significant with a P adj < e-7 (Fig. 4C).These three Reactome pathways were not the top pathways identified in the KB-0742 treatment condition, but they were statistically significant with P adj between e-3 and e-2.These data confirm that the mechanism of action of CDK9 inhibition is to regulate RNA polymerase II mediated transcription as well as other generic transcriptional pathways.1).

Downregulation of MYC and MCL1 is Detected in the Whole Blood of Patients with Enitociclib-treated DH-DLBCL as well as Other MYC+ NHL
To understand whether the downregulation of MYC and MCL1 in vitro translates to patients, the maximum extent of inhibition for MYC mRNA after the first dose of 30 mg enitociclib on C1D1 in patients with DH-DLBCL (n = 10, black lines) and other MYC+ NHL (n = 5, colored lines) whole blood was 89.6% (range, 73.6%-94.8%)and 82.3% (range, 69.9%-90.5%),respectively where maximal inhibition occurred 1-2 hours postdose.On C1D1, maximal MCL mRNA inhibition occurred at 1-2 hours postdose except for patient with the transformed follicular lymphoma, which was observed at 8 hours postdose.

Comparison of DEGs Identified in MYC+ DLBCL Cell Lines to Blood Samples from Patients with Enitociclib-treated MYC+ NHL
Because MYC and MCL mRNA downregulation was observed in cell lines by qPCR in vitro (Fig. 2A and B) and in vivo (Fig. 3D), comparison with RNA-seq in vitro was evaluated.MYC mRNA is statistically significantly downregulated with enitociclib and atuveciclib but was not statistically significant with KB-0472 treatment (Fig. 5A).MCL downregulation is observed but not statistically significant for enitociclib and atuveciclib treatment, whereas MCL mRNA levels are upregulated with KB-0742 treatment.Of note, there are three MCL mRNA splicing variants, of which only one is detected by the qPCR method, but all three are averaged by RNA-seq.RNA-seq analysis from other studies with enitociclib (39) and AZD4573 (40) treatment of CLL and DLBCL cell lines respectively have shown that ZPF36, JUNB, BTG1, and PIM3 are key DEGs with CDK9 inhibition.Herein we show that ZPF36 is significantly downmodulated with enitociclib and atuveciclib treatment and that only 1 μmol/L enitociclib provides statistically significant downregulation of BTG1 and PIM3 (Fig. 5A).DEG analysis by RNA-seq of DH-DLBCL (n = 10) and other MYC+ NHL (n = 5) patient blood samples predose and at multiple timepoints after 30 mg intravenous infusion of enitociclib on C1D1 and C1D15 was performed.PCA demonstrates a robust time-dependent shift in gene expression in patients treated with enitociclib (Supplementary Fig. S1B).The top two DEGs from the 250 nmol/L enitociclib 4 hours in vitro treatment (Fig. 5A, green bars) are confirmed to be reproducibly downregulated after C1D1 and C1D15 in blood  for maximal DEG inhibition suggests a MYC-independent mechanism where novel DEGs identified here PHF23 and TP53RK have maximal inhibition first, followed by known CDK9 targets ZFP36 and PIM3 maximal inhibition, then MYC, MCL1, JUNB, and BTG1 (Supplementary Table S6).

Discussion
Here we investigated the mechanism of action of enitociclib in vitro, in vivo and in blood samples from patients with NHL.Enitociclib was previously shown to drive durable single-agent complete metabolic responses in patients with DH-DLBCL in a phase I trial (29).We show that CDK9  to commit the cell to an apoptotic death (33,34).These preclinical findings were translated into the clinical setting where we show that MYC and MCL mRNA levels are downregulated in the blood of DH-DLBCL but that also this mechanism of action is confirmed in patients with other MYC+ NHL across various indications and not limited to DH-DLBCL.Although the extent or duration of downregulation does not correlate with enitociclib efficacy, the unbound IC 50 of both MYC and MCL mRNA are both clinically achievable with the 30 mg dose, even though the MCL IC 50 is equivalent to the C max which is demonstrated by a 55.5% maximal downregulation of MCL mRNA level in blood of patients postdose.There are limitations to this study, namely the small patient sample size tested and the fact that mRNA levels were measured only from the blood of patients because tumor tissue was not available.A comparison of the pharmacodynamic effect in both blood and tumor tissue samples would provide direct evidence of target knockdown in tumor tissue, but this phase I clinical trial did not mandate pretreatment and posttreatment tumor biopsies.
To understand the transcriptional impact of CDK9 inhibition beyond known targets such as MYC and MCL1, an unbiased transcriptomic analysis has identified DEGs that may be novel CDK9 gene targets in MYC+ DLBCL cell lines.
Treatment with enitociclib using clinically relevant conditions has the expected transcriptional footprint for an inhibitor of RNA polymerase II where 78.94% DEGs are downregulated.In addition to the downregulation of MYC, other genes have been identified as being downregulated by CDK9 inhibition and this observation has been shown in some cases to precede MYC maximal downregulation, as is the case for the top two DEGs in the unbiased analysis, PHF23 and TP53RK.Pathway analysis revealed that RNA transcription pathways are driving the DEGs identified, which is fitting with the proposed mechanism of action.For relative comparison, MYC does not rank in the top DEGs of this study, but the extent of downregulation (−17.108-foldchange) is greater than the novel DEGs identified further strengthening the utility of selective CDK9 inhibitors for targeting MYC+ lymphomas.
In support of the transcriptional shift observed, other transcription factors known to be downregulated by CDK9 inhibition were evaluated in the RNAseq dataset.JUNB, ZFP36, and BTG1 were identified in an RNA-seq analysis of CLL cells treated with enitociclib in vitro (39).We demonstrate that in MYC+ DLBCL cell lines as well as in the blood of patients with DH-DLBCL and other MYC+ NHL, these three transcription factors are also downregulated.Furthermore, in DLBCL cell lines in vitro, PIM3 was identified as a key mediator of epigenetic resistance to another pan-CDK inhibitor, AZD4573 (40).We demonstrate that PIM3 was also downregulated by enitociclib treatment in DLBCL cell lines in vitro and in the blood of patients with DH-DLBCL and other MYC+ NHL.The authors demonstrated that PIM2/3, but not PIM1 inhibition could reverse the resistance (40) yet our data might suggest that rather than a combination of AZD4573 with pan-PIM inhibitor, single agent enitociclib could prevent or treat emergent resistance due to PIM3 overexpression, but confirmation in larger datasets would be required as well as supportive data from corresponding tumor tissue to match observations in blood samples.We which forms fusion oncoprotein with NUP98, which is associated with an aggressive form of AML (44).PHF23 is a negative regulator of autophagy (45) and perhaps explains escape from cell death.The fusion drives overexpression, therefore, downregulation with enitociclib might allow autophagy and provide a therapeutic opportunity for NUP98-PHF23-driven AML that has a poor survival rate.TP53RK has been linked with colorectal cancer metastasis (46) and identified as a target for multiple myeloma (47) and enitociclib may provide an opportunity to target TP53RK.Both PHF23 and TP53RK mRNA downregulation was confirmed by RNA-seq analysis in the blood of enitociclib-treated patients and further studies are warranted to uncover the utility of enitociclib to downregulate these potential cancer targets.

Conclusion
This study demonstrates the biologic advantage of a selective CDK9 inhibitor for the treatment of MYC+ lymphoma preclinical models which is confirmed by analysis of blood samples from patients treated with 30 mg enitociclib i.v.once weekly.Robust transcriptomic down regulation of transcription factors MYC, JUNB, ZFP36, BTG1 as well as novel targets PHF23 and TP53RK was observed with weekly enitociclib dosing while providing a differential decay of prosurvival and antiapoptotic signals.Taken together, our data demonstrate that enitociclib treatment enables durable downregulation of oncogenic protein levels without continuous dosing, resulting in a robust transcriptional shift promoting cancer cell apoptosis and antitumor efficacy.Currently, enitociclib is being evaluated in patients diagnosed with DH-DLBCL and other relapsed/refractory lymphoid malignancies to determine safety and efficacy of enitociclib treatment given as monotherapy or in combination (ClinicalTrials.govIdentifiers: NCT04978779 and NCT05371054).
Two MYC-driven DLBCL cell line models, SU-DHL-4 and SU-DHL-10, were used to evaluate the enitociclib mechanism of action with IC 50 values of 43 nmol/L and 74 nmol/L, respectively.A 0.25 μmol/L concentration [unbound concentration = 167 nmol/L (250 nmol/L X 66.6% (fraction unbound in cell media))] was included in the in vitro study as it provided unbound concentrations comparable C max [unbound C max = 117 nmol/L (1,750 nmol/L (C max ) X 6.65% (fu))] in patients dosed with 30 mg i.v.enitociclib once weekly.In patients with multiple dose data on C1D15 (n = 10), the unbound C max range is 67.5 to 147 nmol/L.As we were investigating the effects of "oncogenic shock" induced by enitociclib, unbound C max was deemed a relevant concentration to target in vitro.Furthermore, the mean t 1/2 as measured in patients treated with multiple doses of enitociclib is 5.56 hours ± 1.93 hours and therefore a 4-hour pretreatment of 250 nmol/L enitociclib was used to model dosing in vitro.The 250 nmol/L in vitro enitociclib concentration used in these experiments is based on the clinical exposure of enitociclib in 15 patients with MYC+ lymphoma; however, this is an estimate and may be superphysiologic.Baseline genomic profiles of RNA-seq analysis demonstrated that both cell lines have comparable MYC transcript levels, despite different MYC DNA status where SU-DHL-4 cell line has MYC gene amplification and SU-DHL-10 has MYC P57T mutation driving MYC overexpression.The levels of MCL mRNA in SU-DHL-10 cells were almost twice as high compared with the SU-DHL-4 cells (Fig.1A).A 4-hour pretreatment with either enitociclib (0.25 or 1 μmol/L), atuveciclib (1 μmol/L), or KB-0742 (1 μmol/L) was washed

FIGURE 2
FIGURE 2 Enitociclib depletes mRNA transcripts, MYC and MCL1; downregulates Myc and Mcl-1 protein levels and activates cell death in vitro MYC (A) and MCL1 (B) mRNA levels were determined using qPCR normalized to 18s rRNA from SU-DHL-10 and SU-DHL-4 DLBCL cells treated with indicated concentration of CDK9 inhibitor for 4 hours.The protein levels of Myc (C) and Mcl-1 (D) pretreatment and at indicated times after treatment washout were quantified relative to GAPDH in SU-DHL-10 and SU-DHL-4 cells.The inset shows Myc protein levels in SU-DHL-10 cells until 8 hours after treatment.E, The protein levels of cPARP is quantified from SU-DHL-10 DLBCL cells pretreatment and at indicated times after treatment.

FIGURE 3
FIGURE 3 Enitociclib treatment results in complete regression of MYC-overexpressing SU-DHL-10 lymphoma growth and mechanism of action confirmed in vivo.A, Growth curves where red arrows indicate dosing days and tumor volumes of SU-DHL-10 tumors treated with either vehicle [30 or 60% PEG400, 10% ethanol (EtOH), water for infusion] or enitociclib at 10 or 15 mg/kg (once weekly, i.v., n = 12/group) for 3 weeks.On days 16 and 20, the T/C ratios for 10 and 15 mg/kg enitociclib were 0.19 and 0.005, respectively.Unpaired t test were performed using comparison with the corresponding vehicle treatment: **, P = 0.011; ***, P < 0.001.B, Body weight changes were observed after application of enitociclib followed by recovery before the next application.C, Phosphorylated RNA polymerase II protein levels at residue Ser2 (p-Ser2) and Ser5 (p-Ser5) were quantified MYC and MCL1 mRNA levels (D) assessed by qPCR and Myc, Mcl-1, cPARP protein levels (E) were quantified from in vivo xenograft of SU-DHL-10 DLBCL cells at predose and indicated times postdose in vivo.Error bars denote SD.

FIGURE 4
FIGURE 4 Enitociclib treatment confers a robust shift in transcriptional activity in MYC+ DLBCL cell lines.A, Comparison of significant DEGs (P adj ≤ 0.05, fold change ≥2) after 4 hours pretreatment of DLBLC cell lines with three CDK9 inhibitors with unsupervised hierarchical clustering based on 250 nmol/L enitociclib treatment.Heat map showing gene intensity per sample relative to the average level across all samples.Individual genes are shown on the Y axis while samples are shown along the X axis.Red and blue cells correspond to higher and lower RNA-seq levels, respectively after 4 hours of treatment.B, List of top DEGs for each treatment ranked by P adj (cutoff 10-7) with overlapping DEGs as indicated by color coding.C, Significant DEGs were analyzed for enrichment of Reactome pathway membership using a hypergeometric test by mapping genes to genes (if appropriate) for each treatment condition and for KB-0742 treated cells, the P adj value cutoff for the pathway of 10-7 was selected for graphical representation because there were 182 significant pathways (30 shown).The three top pathways for enitociclib and atuveciclib are shown for KB-0742 but the P adj values do not meet the cutoff but are included for comparative purposes.
samples of patients with DH-DLBCL and other MYC+ NHL at approximately -1-and -2-fold change from enitociclib predose for PHF23 and TP53RK mR-NAs, respectively (Fig.5B).Known CDK9 target genes from the literature MYC, MCL1, ZFP36, JUNB, BTG1, and PIM3 are shown to compare the extent of downregulation of novel targets PHF23 and TP53RK.Downregulation of transcription factors ZFP and JUNB mRNA levels were > −2 in patients with DH-DLBCL after C1D1 and C1D15 and were approximately −2-fold change after C1D1 and decreased toward −1 after C1D15 in the blood of patients with other MYC+ NHL (n = 3).Similarly, BTG and PIM were also confirmed to be downregulated in the blood of patients treated with enitociclib whereas MCL mRNA fold change was the least robust (Fig.5B).In support of the mechanism of action elucidated herein, MYC fold change downregulation in the blood of patients treated with enitociclib was the most robust finding in this group of genes.The mean fold change mRNA levels of MYC in patients with DH-DLBCL and other MYC+ NHL were −3.45 (SD 0.68) and −2.61 (SD 0.54), respectively after C1D1 dose; and after C1D15 dose, MYC mRNA fold change was −3.38 (SD 1.07) and −1.97 (SD 0.75) for DH-DLBLC and other MYC+ NHL, respectively.These data confirm that robust MYC mRNA downregulation is consistent across in vitro, in vivo and blood samples from patients with NHL treated with enitociclib.The maximal DEG inhibition with enitociclib treatment demonstrates that MYC is the most robust, but the time course

FIGURE 5
FIGURE 5 Comparison of DEGs identified in MYC+ DLBCL cell lines to blood samples from enitociclib-treated patienst with MYC+ NHL.A, Comparison of significant DEGs (P adj ≤ 0.05, fold change ≥2) after at 4 hours pretreatment with 250 nmol/L enitociclib.Heat map showing gene intensity per sample relative to the average level across all samples.Individual genes are shown on the Y axis while samples are shown along the X axis.Red and blue cells correspond to higher and lower RNA-seq levels, respectively after 4 hours of treatment.B, Transcription factors MYC, JUNB, ZFP36 mRNA have statistically significant fold change downregulation with enitociclib treatment in DLBCL cell lines SU-DHL-4 and SU-DHL-10 where other 4 hours treatments do not.PHF23 and TP53RK were the most significantly DEGs with enitociclib treatment at both concentrations.* denotes P adj ≤ 0.05 and fold change ≥2.
further identify two novel targets in this study by RNA-seq of MYC+ DLBCL cell lines treated with 250 nmol/L enitociclib.PHF23 and TP53RK are downregulated and are the two top statistically significant DEGs with robust -10-to -12-fold change in expression postdose (P adj e-12 and e-8), respectively.Histone H3 lysine methylation is a critical component in regulating gene expression, epigenetic states, and cellular identities and is interpreted by conserved modules including plant homeodomain (PHD) fingers of which PHD containing proteins have been deregulated in many types of cancers(43) including PHF23

TABLE 1
Response and pharmacokinetics properties of patients with DH-DLBCL and other patients with MYC+ NHL (n = 15) treated with 30 mg enitociclib i.v.once weekly Dose was lowered from 30 to 22.5 mg (pharmacokinetic data were not included in average). b